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Effects of potassium channel modulators on human detrusor smooth muscle myogenic phasic contractile activity: Potential therapeutic targets for overactive bladder
BASIC SCIENCE
EFFECTS OF POTASSIUM CHANNEL MODULATORS ON HUMAN DETRUSOR SMOOTH MUSCLE MYOGENIC PHASIC CONTRACTILE ACTIVITY: POTENTIAL THERAPEUTIC TARGETS FOR OVERACTIVE BLADDER BENOÎT DARBLADE, DELPHINE BEHR-ROUSSEL, STÉPHANIE OGER, JEAN-PAUL HIEBLE, THIERRY LEBRET, DIANE GORNY, GÉRARD BENOIT, LAURENT ALEXANDRE, AND FRANÇOIS GIULIANO
ABSTRACT Objectives. Increased urinary bladder detrusor smooth muscle phasic contractility has been suggested to be associated with idiopathic bladder overactivity (OAB). We examined the role of voltage-dependent L-type calcium channels, adenosine triphosphate-sensitive potassium (KATP) channels, and calcium-activated potassium (BKCa and SKCa) channels in the regulation of human detrusor phasic contractile activity. Methods. Isolated human bladder strip phasic contractions were measured and quantified as the mean area under the force-time curve, amplitude, and frequency of phasic contractions in 22 bladder samples. Results. Human detrusor strips displayed myogenic phasic contractions in the presence of atropine (10⫺6 M), phentolamine (10⫺6 M), propranolol (10⫺6 M), suramin (10⫺5 M), and tetrodotoxin (10⫺6 M). The L-type calcium channel inhibitor nifedipine (300 nM) abolished the contractile activity. Blockade of KATP channels by glibenclamide (1 and 10 M) did not alter myogenic contractions. In contrast, the KATP channel opener pinacidil (10 M) markedly inhibited phasic contractility. Iberiotoxin (100 nM) and apamin (100 nM), potent and selective inhibitors of BKCa and SKCa channels, respectively, significantly increased the area under the force-time curve and the amplitude of contractions. Conclusions. Phasic contractions of human detrusor are dependent on calcium entry through L-type calcium channels. BKCa and SKCa channels play a key role in the modulation of human detrusor smooth muscle phasic contractility. Furthermore, these observations support the concept that increasing conductance through KATP, BKCa, and SKCa channels may represent attractive pharmacologic targets for decreasing phasic contractions of detrusor smooth muscle in OAB. UROLOGY 68: 442–448, 2006. © 2006 Elsevier Inc.
O
veractive bladder (OAB) is a common pathologic condition resulting from a neurogenic or nonneurogenic etiology that can be related to outflow obstruction or aging, but most cases are idiopathic.1 Myogenic changes (ie, modification in deFrom Pelvipharm, Domaine CNRS, Gif-sur-Yvette, France; Department of Urology Research, GlaxoSmithKline, King of Prussia, Pennsylvania; Department of Urology, Hôpital Foch, Suresnes, France; Department of Urology, CHU de Bicêtre, Le Kremlin Bicêtre, France; and Neuro-Urology Unit, Department of Neurological Rehabilitation, Raymond Poincaré Hospital, Garches, France Reprint requests: François Giuliano, M.D., Neuro-Urology Unit, Department of Neurological Rehabilitation, Raymond Poincaré Hospital, 104 bd Raymond Poincaré, Garches 92380, France. E-mail: [email protected] Submitted: November 10, 2005, accepted (with revisions): March 21, 2006 © 2006 ELSEVIER INC. 442
ALL RIGHTS RESERVED
trusor smooth muscle properties) may be a prerequisite for the occurrence of idiopathic detrusor overactivity.2 Detrusor develops spontaneous phasic contractions that can be observed in humans and animals in vivo from cystometrography3,4 and in vitro from detrusor isolated strips.5,6 Under physiologic conditions, these contractions are not responsible for any increase in intravesical pressure, because the synchronized contractions of the whole bladder occurring during the filling and voiding phases are under control of the autonomic innervation. Alterations in the electrical properties of detrusor cells may result in spontaneous synchronized contractions of smooth muscle fibers bundles leading to bladder overactivity.6 –9 In this regard, spontaneous detrusor activity has been reported to be impaired in bladder strips obtained 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.03.039
from patients with idiopathic OAB9 and in animal models of OAB caused by partial bladder outlet obstruction.3,10 Therefore, a better understanding of the regulation of myogenic spontaneous bladder contractions may contribute to the identification of new therapeutic targets in the treatment of OAB, because the current standard pharmacologic treatment (ie, muscarinic antagonists) has limitations owing to limited efficacy and side effects.11 In vitro studies of isolated bladder strips from various animal species12–15 have suggested that phasic contractile activity is myogenic in nature. Indeed, independently of any neural stimulation, detrusor cells display spontaneous rhythmic action potentials that are responsible for extracellular Ca2⫹ entry through dihydropyridine-sensitive, voltage-dependent L-type Ca2⫹ channels and successive Ca2⫹ release through ryanodine-sensitive channels of the sarcoplasmic reticulum.13,16 The repolarization of the upstroke of the action potential caused by Ca2⫹ entry through L-type Ca2⫹ channels depends on activation of different types of K⫹ channels. Involvement of Ca2⫹-activated K⫹ channels (ie, iberiotoxin-sensitive large-conductance K⫹ channels [BKCa] and apamin-sensitive smallconductance K⫹ channels [SKCa]) and adenosine triphosphatase-dependent K⫹ channels (KATP) in the negative-feedback modulation of phasic detrusor smooth muscle contractions has been functionally evaluated in animal models.13–15 Despite the likely functional importance of bladder phasic contractions in OAB, little is known about the potential influence of pharmacologic modulators of K⫹ channels on human detrusor phasic contractile activity. The present study was conducted to evaluate the consequences of KCa and KATP channel modulators on human detrusor phasic contractions. MATERIAL AND METHODS BLADDER STRIP PREPARATION Twenty-two samples of human bladders were obtained from 22 patients undergoing cystectomy for bladder cancer. The bladder samples were collected with patient-informed consent. Only donors with no known OAB according to their medical chart were solicited. A normal bladder dome sample (ie, with no macroscopic tumoral tissue) was harvested and stored at 4°C in Krebs-4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer (millimolar composition: NaCl 118.0, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 4.2, glucose 11.1, and HEPES 20.8; pH 7.4) containing penicillin (100 UI/mL) and streptomycin (0.1 mg/mL). Detrusor strips (8 mm ⫻ 4 mm; mean wet weight 15.21 ⫾ 4.97 mg) were prepared by removing the serosal and mucosal layers.
IN VITRO HUMAN BLADDER CONTRACTILE STUDIES The strips were mounted isometrically at a resting tension of 1 g in 5-mL organ baths filled with Krebs-HEPES buffer UROLOGY 68 (2), 2006
maintained at 37°C and bubbled with 95% dioxygen and 5% carbon dioxide. The strips were connected to force transducers and the tension changes recorded using MacLab, version 8, and Chart, version 5, software (AD Instruments). The strips were equilibrated for 90 minutes. Spontaneous phasic contractions were recorded in 40% of all the bladder strips during this period. The mean resting tone of the strips was 0.425 ⫾ 0.6 g at the end of the equilibration period and spontaneous fused tetanic contractions were very rarely observed. They were exposed to 10 mM KCl and allowed to equilibrate for 30 minutes to enhance the development of phasic contractions. Strips showing unstable responses or that did not display regular contractions after 30 minutes of incubation with KCl were discarded (about 25%).
STATISTICAL ANALYSIS Phasic contractile activity was quantified by calculating the area under the force-time curve (AUC) using the low points of the phasic contractions as the baseline, maximal amplitude, and frequency of phasic contractions, as described previously.14,15 Analyses were performed with Elphy, version 3.0.0.45, software (CNRS-UNIC, France). The AUC, amplitude, and frequency are expressed as the percentage in the change of initial values measured before exposure to every tested pharmacologic agent. Data are expressed as the mean ⫾ SEM. Comparisons were performed using Student’s t test. P values less than 0.05 were considered significant (GraphPad Prism, version 4.02).
DRUGS AND CHEMICALS All drugs and chemicals were purchased from Sigma Chemical (St. Louis, Mo). Nifedipine, glibenclamide, and pinacidil were prepared in dimethyl sulfoxide, with a final concentration of 0.01% in the organ bath, except for glibenclamide 10 M, for which the final concentration was 0.1%. Apamin was prepared in acetic acid (0.05 mM final concentration in the organ bath). At the concentrations used, dimethyl sulfoxide or acetic acid had no effect on detrusor activity. All other drugs were prepared in distilled water.
RESULTS MYOGENIC ORIGIN OF PHASIC CONTRACTILE ACTIVITY To verify that phasic contractions enhanced by 10 mM KCl were not caused by neurotransmitters released from autonomic nerves, the bladder strips were exposed to a drug cocktail containing blockers for known transmitter receptors in the bladder wall, including the muscarinic antagonist atropine (10⫺6 M), alpha-adrenergic antagonist phentolamine (10⫺6 M), beta-adrenergic antagonist propranolol (10⫺6 M), purinergic antagonist suramin (10⫺5 M), and neuronal sodium channel blocker tetrodotoxin (10⫺6 M).13,14 Phasic contractions persisted in the presence of the drug cocktail in terms of the AUC, amplitude, or frequency, suggesting a myogenic origin for these contractions (data not shown). ROLE OF L-TYPE Ca2ⴙ CHANNELS The involvement of Ca2⫹ influx through L-type Ca2⫹ channels in the generation of phasic contractions was evaluated by exposure to the L-type Ca2⫹ channel antagonist nifedipine. Phasic contractile activity was reduced to 4.9% ⫾ 3.9% of the initial 443
FIGURE 1. Effect of nifedipine on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip exposed to nifedipine (300 nM). (B) Effects of nifedipine or vehicle on AUC of time-force curve of phasic contractions. Data are mean ⫾ SEM of five and six bladder samples for nifedipine-treated and vehicle-treated conditions, respectively. Mean responses expressed as percentage of initial values measured before exposure to either nifedipine or vehicle . ***P ⬍0.001, nifedipine versus vehicle.
activity in the presence of nifedipine (300 nM) versus 87.0% ⫾ 10.0% of the initial AUC in vehicle condition (AUC, nifedipine versus vehicle, P ⬍0.001; Fig. 1). ROLE OF KATP AND KCa CHANNELS The implication of the KATP channel in the regulation of phasic contractility was examined by incubating the strips with the KATP channel inhibitor glibenclamide and the KATP channel opener pinacidil. Glibenclamide (1 and 10 M) did not elicit any significant changes in phasic contractile activity (Fig. 2A). In particular, the variations in the AUC measured after exposure to 1 or 10 M glibenclamide were not significantly different from those observed after exposure to vehicle at similar concentrations (116.7% ⫾ 18.0% versus 84.1% ⫾ 37.6% and 131.1% ⫾ 81.0% versus 72.6% ⫾43.9% of the initial AUC; Fig. 2B). Pinacidil (10 M) induced suppression of phasic contractile activity (Fig. 3). It reduced the AUC to 91.4% ⫾ 3.1% versus 15.3% ⫾ 37.6% with vehicle (P ⬍0.05), amplitude by 80.6% ⫾ 10.2% versus 4.2% ⫾ 19.0% with vehicle (P ⬍0.01), and frequency of the phasic contractions by 63.1% ⫾ 14.1% versus 36.6% ⫾ 20.1% with vehicle (P ⬍0.05). This inhibitory effect was reversed by the addition of 10 M glibenclamide. This recovery effect was significant on the AUC and the frequency of phasic contractions (P ⬍0.05). In addition, glibenclamide significantly enhanced the AUC and the amplitude of phasic contractile activity after exposure to pinacidil compared with vehicle (P ⬍0.05). The role of KCa channels was evaluated using the selective BKCa channel inhibitor iberiotoxin and the selective SKCa channel inhibitor apamin. Iberiotoxin (100 nM) elicited a significant increase in both the 444
initial AUC (245.3 ⫾ 59.9% versus ⫺2.7% ⫾ 12.9% with vehicle, P ⬍0.05) and the amplitude of the phasic contractions (155.6% ⫾ 47.4% versus 23.3% ⫾ 24.7% with vehicle, P ⬍0.05, Fig. 4A,C). Apamin (100 nM) significantly augmented the AUC by 284.9% ⫾ 109.4% and the amplitude of contractions by 294.9% ⫾ 94.6% versus vehicle (P ⬍0.05, Fig. 4B,C). COMMENT A pharmacologic and functional approach was used to examine the role of various ion channels in the regulation of human detrusor phasic contractile activity, including the L-type Ca2⫹, Ca2⫹-activated K⫹ (ie, BKCa and SKCa), and KATP channels. Spontaneous contractile activity of the bladder strips was recorded in our experiments in 40% of all strips during the equilibration period. These results are different from those of Mills et al.,6 in which 69% of the bladder strips from cadaveric controls without a previous history of urologic dysfunction had spontaneous contractile activity. This variability in the results could be explained by several factors such as the median age of the patients in our study, which was greater than that in the study by Mills et al.6 (63.3 versus 50.5 years, respectively) or the proportion of female versus male patients (5 women and 17 men in our study versus 8 women and 6 men in their study). In contrast, the mean resting tone of the spontaneous contractions and the occurrence of fused tetanic contractions were similar between the two studies. Although the myogenic nature of bladder phasic contractile activity has been clearly established in guinea pig13,14 and pig15 isolated bladder preparaUROLOGY 68 (2), 2006
FIGURE 2. Effect of glibenclamide on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip exposed to glibenclamide (1 and 10 M). (B) Effects of glibenclamide or vehicle on AUC of time-force curve, amplitude, and frequency of phasic contractions. Data are mean ⫾ SEM of three and five bladder samples for glibenclamide-treated and vehicle-treated conditions, respectively. Mean responses expressed as percentage of initial values measured before exposure to drug. P ⫽ NS, glibenclamide versus vehicle.
tions and shown in human detrusor in the present study, the mechanisms responsible for the occurrence of phasic mechanical activity have not yet been established. These phasic spontaneous events seem to be closely associated with spontaneous action potential. Spontaneous action potentials and associated calcium waves occur almost simultaneously along the boundary of bladder smooth muscle bundles and then propagate to the other boundary, probably through gap junctions.17 The investigation of electrical properties of human detrusor has revealed that calcium and potassium channels play an important role in the regulation of electrical activity of detrusor smooth muscle fibers.17 Thus, one may speculate that the participation of these channels in the regulation of human phasic contractile activity has been strongly suggested but never been clearly demonstrated. In human detrusor, L-type Ca2⫹ channels have been identified.18 Moreover, in guinea pig and pig isolated bladder strips, spontaneous phasic contractions are sensitive to the L-type Ca2⫹ channel inhibitors nisoldipine13,14 and nifedipine,15 respectively. Consistent with these data, we found that UROLOGY 68 (2), 2006
nifedipine suppressed human detrusor phasic contractions, suggesting that human detrusor phasic contractions also require extracellular Ca2⫹ entry through L-type Ca2⫹ channels. First, we investigated the participation of KATP channels in the regulation of human detrusor phasic contractions, because their participation has already been established in other animal species.15,19 We found no effect of the KATP channel inhibitor, glibenclamide, on phasic contractile activity, in accordance with data obtained from guinea pig19 and pig15 studies. This could suggest that KATP channels are closed and thus do not participate in the regulation of phasic contractions under basal conditions. KATP channels may rather be involved in the regulation of detrusor agonist-induced relaxation.20,21 In contrast, the KATP channel opener pinacidil dramatically reduced phasic contractile activity, and this effect was reversed by glibenclamide. Thus, one may speculate that the activation of KATP channels by KATP channel openers such as pinacidil22 or cromakalim15,23–25 may be sufficient to provoke membrane hyperpolarization, thereby altering smooth muscle cell contrac445
FIGURE 3. Effect of pinacidil and glibenclamide on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip displaying effect of pinacidil (10 M) on phasic contractions and restoring effect of glibenclamide (10 M). (B) Successive effects of pinacidil, glibenclamide, or their respective vehicles on AUC of time-force curve, amplitude, and frequency of phasic contractions. Data are mean ⫾ SEM of six and five bladder samples for drug-treated and vehicle conditions, respectively. Mean responses expressed as percentages of initial values measured before exposure to drugs. *P ⬍0.05, **P ⬍0.01, pinacidil versus vehicle; # P ⬍0.05, pinacidil versus glibenclamide; §P ⬍0.05, glibenclamide versus vehicle.
tility and disturbing the initiation of phasic contractions.15,26 Second, we also explored the participation of KCa (BK and SK) channels. The potent and selective blockers of the BKCa channel iberiotoxin and of SK2 and SK3 subtypes of the SKCa channel apamin markedly enhanced in vitro human bladder phasic contractions. Similar observations have been previously reported in isolated pig bladder.15 In addition, iberiotoxin has been reported to induce membrane depolarization and increase the frequency, amplitude, and duration of action potentials in guinea pig detrusor strips.27 Thus, the increase in phasic contractions induced by iberiotoxin or apamin we observed may have resulted from an increase of action potential amplitudes and/or frequencies and an enhancement of entry of Ca2⫹ through L-type Ca2⫹ channels. Also, studies with transgenic mice demonstrated a central role of BKCa and SKCa channels in urinary bladder function, particularly in spontaneous contractile activity.28,29 Thus, our results demonstrate the physiologic involvement of BKCa, SK2, and/or SK3 channels in the regulation of phasic contrac446
tile activity of the human detrusor. They could play prominent roles as negative-feedback elements to limit extracellular Ca2⫹ influx-mediated human detrusor phasic contractility. Additional studies investigating the effects of selective BKCa or SKCa channel openers are thus needed to better understand these mechanisms. Such selective openers might be expected to decrease human detrusor activity. Commercially available tools lack the selectivity required to test this hypothesis. CONCLUSIONS Pilot clinical studies performed in patients with OAB that tested first-generation K⫹ channel openers have revealed that these compounds may improve symptoms of urinary frequency and increase the mean voided volume; however, the benefit was limited by cardiovascular side effects.30,31 In the present study, we demonstrated that KATP and KCa channels participate in the regulation of human bladder myogenic phasic contractility. If myogenic spontaneous bladder contractions are involved in the origin of OAB, as previously suggested,2,8 uroUROLOGY 68 (2), 2006
FIGURE 4. Effect of iberiotoxin and apamin on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip exposed to iberiotoxin (100 nM). (B) Original tension recording developed by one detrusor strip exposed to apamin (100 nM). (C) Effects of iberiotoxin or apamin or vehicle on AUC of time-force curve, amplitude, and frequency of phasic contractions. Data are mean ⫾ SEM of five, six, and five bladder samples for iberiotoxin-treated, apamin-treated, and vehicle-treated conditions, respectively. Mean responses expressed as percentages of initial values measured before exposure to iberiotoxin, apamin, or vehicle. *P ⬍0.05, iberiotoxin versus vehicle and apamin versus vehicle.
selective potassium channel openers, acting on detrusor smooth muscle with an acceptable cardiovascular safety profile, may be a promising pharmacologic strategy for the treatment of OAB. REFERENCES 1. Stewart WF, Van Rooyen JB, Cundiff GW, et al: Prevalence and burden of overactive bladder in the United States. World J Urol 20: 327–336, 2003. UROLOGY 68 (2), 2006
2. Brading AF: A myogenic basis for the overactive bladder. Urology 50(6A suppl): 57– 67, 1997. 3. Persson K, Pandita RK, Waldeck K, et al: Angiotensin II and bladder obstruction in the rat: influence on hypertrophic growth and contractility. Am J Physiol 271(5 Pt 2): R1186 – R1192, 1996. 4. Robertson AS: Behaviour of the human bladder during natural filling: the Newcastle experience of ambulatory monitoring and conventional artificial filling cystometry. Scand J Urol Nephrol Suppl 201: 19 –24, 1999. 447
5. Sibley GN: A comparison of spontaneous and nervemediated activity in bladder muscle from man, pig and rabbit. J Physiol 354: 431– 443, 1984. 6. Mills IW, Greenland JE, McMurray G, et al: Studies of the pathophysiology of idiopathic detrusor instability: the physiological properties of the detrusor smooth muscle and its pattern of innervation. J Urol 163: 646 – 651, 2000. 7. Brading AF, and Turner WH: The unstable bladder: towards a common mechanism. Br J Urol 73: 3– 8, 1994. 8. Fry CH, Sui GP, Severs NJ, et al: Spontaneous activity and electrical coupling in human detrusor smooth muscle: implications for detrusor overactivity? Urology 63(3 suppl 1): 3–10, 2004. 9. Kinder RB, and Mundy AR: Pathophysiology of idiopathic detrusor instability and detrusor hyper-reflexia: an in vitro study of human detrusor muscle. Br J Urol 60: 509 –515, 1987. 10. Sibley GN: An experimental model of detrusor instability in the obstructed pig. Br J Urol 57: 292–298, 1985. 11. Andersson KE: Antimuscarinics for treatment of overactive bladder. Lancet Neurol 3: 46 –53, 2004. 12. Liu SP, Volfson I, Horan P, et al: Effects of hypoxia, calcium, carbachol, atropine and tetrodotoxin on the filling of the in-vitro rabbit whole bladder. J Urol 160(3 Pt 1): 913–919, 1998. 13. Herrera GM, Heppner TJ, and Nelson MT: Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels. Am J Physiol Regul Integr Comp Physiol 279: R60 –R68, 2000. 14. Imai T, Okamoto T, Yamamoto Y, et al: Effects of different types of K⫹ channel modulators on the spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle. Acta Physiol Scand 173: 323–333, 2001. 15. Buckner SA, Milicic I, Daza AV, et al: Spontaneous phasic activity of the pig urinary bladder smooth muscle: characteristics and sensitivity to potassium channel modulators. Br J Pharmacol 135: 639 – 648, 2002. 16. Brading AF: Ion channels and control of contractile activity in urinary bladder smooth muscle. Jpn J Pharmacol 58(suppl 2): 120P–127P, 1992. 17. Hashitani H, and Brading AF: Electrical properties of detrusor smooth muscles from the pig and human urinary bladder. Br J Pharmacol 140: 146 –158, 2003. 18. Sui GP, Wu C, and Fry CH: A description of Ca2⫹ channels in human detrusor smooth muscle. BJU Int 92: 476 – 482, 2003. 19. Imai T, Okamoto T, Yamamoto Y, et al: Effects of different types of K⫹ channel modulators on the spontaneous
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myogenic contraction of guinea-pig urinary bladder smooth muscle. Acta Physiol Scand 173: 323–333, 2001. 20. Hudman D, Elliott RA, and Norman RI: K(ATP) channels mediate the beta(2)-adrenoceptor agonist-induced relaxation of rat detrusor muscle. Eur J Pharmacol 397: 169 –176, 2000. 21. Deka DK, and Brading AF: Nitric oxide activates glibenclamide-sensitive K⫹ channels in urinary bladder myocytes through a c-GMP-dependent mechanism. Eur J Pharmacol 492: 13–19, 2004. 22. Malmgren A, Andersson KE, Sjogren C, et al: Effects of pinacidil and cromakalim (BRL 34915) on bladder function in rats with detrusor instability. J Urol 142: 1134 –1138, 1989. 23. Foster CD, Speakman MJ, Fujii K, et al: The effects of cromakalim on the detrusor muscle of human and pig urinary bladder. Br J Urol 63: 284 –294, 1989. 24. Fujii K, Foster CD, Brading AF, et al: Potassium channel blockers and the effects of cromakalim on the smooth muscle of the guinea-pig bladder. Br J Pharmacol 99: 779 – 785, 1990. 25. Wammack R, Jahnel U, Nawrath H, et al: Mechanical and electrophysiological effects of cromakalim on the human urinary bladder. Eur Urol 26: 176 –181, 1994. 26. Shieh CC, Feng J, Buckner SA, et al: Functional implication of spare ATP-sensitive K(⫹) channels in bladder smooth muscle cells. J Pharmacol Exp Ther 296: 669 – 675, 2001. 27. Heppner TJ, Bonev AD, and Nelson MT: Ca(2⫹)-activated K⫹ channels regulate action potential repolarization in urinary bladder smooth muscle. Am J Physiol 273(1 Pt 1): C110 –C117, 1997. 28. Meredith AL, Thorneloe KS, Werner ME, et al: Overactive bladder and incontinence in the absence of the BK large conductance Ca2⫹-activated K⫹ channel. J Biol Chem 279: 36746 –36752, 2004. 29. Herrera GM, Pozo MJ, Zvara P, et al: Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel. J Physiol 551(Pt 3): 893–903, 2003. 30. Nurse DE, Restorick JM, and Mundy AR: The effect of cromakalim on the normal and hyper-reflexic human detrusor muscle. Br J Urol 68: 27–31, 1991. 31. Komersova K, Rogerson JW, Conway EL, et al: The effect of levcromakalim (BRL 38227) on bladder function in patients with high spinal cord lesions. Br J Clin Pharmacol 39: 207–209, 1995.
UROLOGY 68 (2), 2006
EFFECTS OF POTASSIUM CHANNEL MODULATORS ON HUMAN DETRUSOR SMOOTH MUSCLE MYOGENIC PHASIC CONTRACTILE ACTIVITY: POTENTIAL THERAPEUTIC TARGETS FOR OVERACTIVE BLADDER BENOÎT DARBLADE, DELPHINE BEHR-ROUSSEL, STÉPHANIE OGER, JEAN-PAUL HIEBLE, THIERRY LEBRET, DIANE GORNY, GÉRARD BENOIT, LAURENT ALEXANDRE, AND FRANÇOIS GIULIANO
ABSTRACT Objectives. Increased urinary bladder detrusor smooth muscle phasic contractility has been suggested to be associated with idiopathic bladder overactivity (OAB). We examined the role of voltage-dependent L-type calcium channels, adenosine triphosphate-sensitive potassium (KATP) channels, and calcium-activated potassium (BKCa and SKCa) channels in the regulation of human detrusor phasic contractile activity. Methods. Isolated human bladder strip phasic contractions were measured and quantified as the mean area under the force-time curve, amplitude, and frequency of phasic contractions in 22 bladder samples. Results. Human detrusor strips displayed myogenic phasic contractions in the presence of atropine (10⫺6 M), phentolamine (10⫺6 M), propranolol (10⫺6 M), suramin (10⫺5 M), and tetrodotoxin (10⫺6 M). The L-type calcium channel inhibitor nifedipine (300 nM) abolished the contractile activity. Blockade of KATP channels by glibenclamide (1 and 10 M) did not alter myogenic contractions. In contrast, the KATP channel opener pinacidil (10 M) markedly inhibited phasic contractility. Iberiotoxin (100 nM) and apamin (100 nM), potent and selective inhibitors of BKCa and SKCa channels, respectively, significantly increased the area under the force-time curve and the amplitude of contractions. Conclusions. Phasic contractions of human detrusor are dependent on calcium entry through L-type calcium channels. BKCa and SKCa channels play a key role in the modulation of human detrusor smooth muscle phasic contractility. Furthermore, these observations support the concept that increasing conductance through KATP, BKCa, and SKCa channels may represent attractive pharmacologic targets for decreasing phasic contractions of detrusor smooth muscle in OAB. UROLOGY 68: 442–448, 2006. © 2006 Elsevier Inc.
O
veractive bladder (OAB) is a common pathologic condition resulting from a neurogenic or nonneurogenic etiology that can be related to outflow obstruction or aging, but most cases are idiopathic.1 Myogenic changes (ie, modification in deFrom Pelvipharm, Domaine CNRS, Gif-sur-Yvette, France; Department of Urology Research, GlaxoSmithKline, King of Prussia, Pennsylvania; Department of Urology, Hôpital Foch, Suresnes, France; Department of Urology, CHU de Bicêtre, Le Kremlin Bicêtre, France; and Neuro-Urology Unit, Department of Neurological Rehabilitation, Raymond Poincaré Hospital, Garches, France Reprint requests: François Giuliano, M.D., Neuro-Urology Unit, Department of Neurological Rehabilitation, Raymond Poincaré Hospital, 104 bd Raymond Poincaré, Garches 92380, France. E-mail: [email protected] Submitted: November 10, 2005, accepted (with revisions): March 21, 2006 © 2006 ELSEVIER INC. 442
ALL RIGHTS RESERVED
trusor smooth muscle properties) may be a prerequisite for the occurrence of idiopathic detrusor overactivity.2 Detrusor develops spontaneous phasic contractions that can be observed in humans and animals in vivo from cystometrography3,4 and in vitro from detrusor isolated strips.5,6 Under physiologic conditions, these contractions are not responsible for any increase in intravesical pressure, because the synchronized contractions of the whole bladder occurring during the filling and voiding phases are under control of the autonomic innervation. Alterations in the electrical properties of detrusor cells may result in spontaneous synchronized contractions of smooth muscle fibers bundles leading to bladder overactivity.6 –9 In this regard, spontaneous detrusor activity has been reported to be impaired in bladder strips obtained 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.03.039
from patients with idiopathic OAB9 and in animal models of OAB caused by partial bladder outlet obstruction.3,10 Therefore, a better understanding of the regulation of myogenic spontaneous bladder contractions may contribute to the identification of new therapeutic targets in the treatment of OAB, because the current standard pharmacologic treatment (ie, muscarinic antagonists) has limitations owing to limited efficacy and side effects.11 In vitro studies of isolated bladder strips from various animal species12–15 have suggested that phasic contractile activity is myogenic in nature. Indeed, independently of any neural stimulation, detrusor cells display spontaneous rhythmic action potentials that are responsible for extracellular Ca2⫹ entry through dihydropyridine-sensitive, voltage-dependent L-type Ca2⫹ channels and successive Ca2⫹ release through ryanodine-sensitive channels of the sarcoplasmic reticulum.13,16 The repolarization of the upstroke of the action potential caused by Ca2⫹ entry through L-type Ca2⫹ channels depends on activation of different types of K⫹ channels. Involvement of Ca2⫹-activated K⫹ channels (ie, iberiotoxin-sensitive large-conductance K⫹ channels [BKCa] and apamin-sensitive smallconductance K⫹ channels [SKCa]) and adenosine triphosphatase-dependent K⫹ channels (KATP) in the negative-feedback modulation of phasic detrusor smooth muscle contractions has been functionally evaluated in animal models.13–15 Despite the likely functional importance of bladder phasic contractions in OAB, little is known about the potential influence of pharmacologic modulators of K⫹ channels on human detrusor phasic contractile activity. The present study was conducted to evaluate the consequences of KCa and KATP channel modulators on human detrusor phasic contractions. MATERIAL AND METHODS BLADDER STRIP PREPARATION Twenty-two samples of human bladders were obtained from 22 patients undergoing cystectomy for bladder cancer. The bladder samples were collected with patient-informed consent. Only donors with no known OAB according to their medical chart were solicited. A normal bladder dome sample (ie, with no macroscopic tumoral tissue) was harvested and stored at 4°C in Krebs-4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer (millimolar composition: NaCl 118.0, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 4.2, glucose 11.1, and HEPES 20.8; pH 7.4) containing penicillin (100 UI/mL) and streptomycin (0.1 mg/mL). Detrusor strips (8 mm ⫻ 4 mm; mean wet weight 15.21 ⫾ 4.97 mg) were prepared by removing the serosal and mucosal layers.
IN VITRO HUMAN BLADDER CONTRACTILE STUDIES The strips were mounted isometrically at a resting tension of 1 g in 5-mL organ baths filled with Krebs-HEPES buffer UROLOGY 68 (2), 2006
maintained at 37°C and bubbled with 95% dioxygen and 5% carbon dioxide. The strips were connected to force transducers and the tension changes recorded using MacLab, version 8, and Chart, version 5, software (AD Instruments). The strips were equilibrated for 90 minutes. Spontaneous phasic contractions were recorded in 40% of all the bladder strips during this period. The mean resting tone of the strips was 0.425 ⫾ 0.6 g at the end of the equilibration period and spontaneous fused tetanic contractions were very rarely observed. They were exposed to 10 mM KCl and allowed to equilibrate for 30 minutes to enhance the development of phasic contractions. Strips showing unstable responses or that did not display regular contractions after 30 minutes of incubation with KCl were discarded (about 25%).
STATISTICAL ANALYSIS Phasic contractile activity was quantified by calculating the area under the force-time curve (AUC) using the low points of the phasic contractions as the baseline, maximal amplitude, and frequency of phasic contractions, as described previously.14,15 Analyses were performed with Elphy, version 3.0.0.45, software (CNRS-UNIC, France). The AUC, amplitude, and frequency are expressed as the percentage in the change of initial values measured before exposure to every tested pharmacologic agent. Data are expressed as the mean ⫾ SEM. Comparisons were performed using Student’s t test. P values less than 0.05 were considered significant (GraphPad Prism, version 4.02).
DRUGS AND CHEMICALS All drugs and chemicals were purchased from Sigma Chemical (St. Louis, Mo). Nifedipine, glibenclamide, and pinacidil were prepared in dimethyl sulfoxide, with a final concentration of 0.01% in the organ bath, except for glibenclamide 10 M, for which the final concentration was 0.1%. Apamin was prepared in acetic acid (0.05 mM final concentration in the organ bath). At the concentrations used, dimethyl sulfoxide or acetic acid had no effect on detrusor activity. All other drugs were prepared in distilled water.
RESULTS MYOGENIC ORIGIN OF PHASIC CONTRACTILE ACTIVITY To verify that phasic contractions enhanced by 10 mM KCl were not caused by neurotransmitters released from autonomic nerves, the bladder strips were exposed to a drug cocktail containing blockers for known transmitter receptors in the bladder wall, including the muscarinic antagonist atropine (10⫺6 M), alpha-adrenergic antagonist phentolamine (10⫺6 M), beta-adrenergic antagonist propranolol (10⫺6 M), purinergic antagonist suramin (10⫺5 M), and neuronal sodium channel blocker tetrodotoxin (10⫺6 M).13,14 Phasic contractions persisted in the presence of the drug cocktail in terms of the AUC, amplitude, or frequency, suggesting a myogenic origin for these contractions (data not shown). ROLE OF L-TYPE Ca2ⴙ CHANNELS The involvement of Ca2⫹ influx through L-type Ca2⫹ channels in the generation of phasic contractions was evaluated by exposure to the L-type Ca2⫹ channel antagonist nifedipine. Phasic contractile activity was reduced to 4.9% ⫾ 3.9% of the initial 443
FIGURE 1. Effect of nifedipine on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip exposed to nifedipine (300 nM). (B) Effects of nifedipine or vehicle on AUC of time-force curve of phasic contractions. Data are mean ⫾ SEM of five and six bladder samples for nifedipine-treated and vehicle-treated conditions, respectively. Mean responses expressed as percentage of initial values measured before exposure to either nifedipine or vehicle . ***P ⬍0.001, nifedipine versus vehicle.
activity in the presence of nifedipine (300 nM) versus 87.0% ⫾ 10.0% of the initial AUC in vehicle condition (AUC, nifedipine versus vehicle, P ⬍0.001; Fig. 1). ROLE OF KATP AND KCa CHANNELS The implication of the KATP channel in the regulation of phasic contractility was examined by incubating the strips with the KATP channel inhibitor glibenclamide and the KATP channel opener pinacidil. Glibenclamide (1 and 10 M) did not elicit any significant changes in phasic contractile activity (Fig. 2A). In particular, the variations in the AUC measured after exposure to 1 or 10 M glibenclamide were not significantly different from those observed after exposure to vehicle at similar concentrations (116.7% ⫾ 18.0% versus 84.1% ⫾ 37.6% and 131.1% ⫾ 81.0% versus 72.6% ⫾43.9% of the initial AUC; Fig. 2B). Pinacidil (10 M) induced suppression of phasic contractile activity (Fig. 3). It reduced the AUC to 91.4% ⫾ 3.1% versus 15.3% ⫾ 37.6% with vehicle (P ⬍0.05), amplitude by 80.6% ⫾ 10.2% versus 4.2% ⫾ 19.0% with vehicle (P ⬍0.01), and frequency of the phasic contractions by 63.1% ⫾ 14.1% versus 36.6% ⫾ 20.1% with vehicle (P ⬍0.05). This inhibitory effect was reversed by the addition of 10 M glibenclamide. This recovery effect was significant on the AUC and the frequency of phasic contractions (P ⬍0.05). In addition, glibenclamide significantly enhanced the AUC and the amplitude of phasic contractile activity after exposure to pinacidil compared with vehicle (P ⬍0.05). The role of KCa channels was evaluated using the selective BKCa channel inhibitor iberiotoxin and the selective SKCa channel inhibitor apamin. Iberiotoxin (100 nM) elicited a significant increase in both the 444
initial AUC (245.3 ⫾ 59.9% versus ⫺2.7% ⫾ 12.9% with vehicle, P ⬍0.05) and the amplitude of the phasic contractions (155.6% ⫾ 47.4% versus 23.3% ⫾ 24.7% with vehicle, P ⬍0.05, Fig. 4A,C). Apamin (100 nM) significantly augmented the AUC by 284.9% ⫾ 109.4% and the amplitude of contractions by 294.9% ⫾ 94.6% versus vehicle (P ⬍0.05, Fig. 4B,C). COMMENT A pharmacologic and functional approach was used to examine the role of various ion channels in the regulation of human detrusor phasic contractile activity, including the L-type Ca2⫹, Ca2⫹-activated K⫹ (ie, BKCa and SKCa), and KATP channels. Spontaneous contractile activity of the bladder strips was recorded in our experiments in 40% of all strips during the equilibration period. These results are different from those of Mills et al.,6 in which 69% of the bladder strips from cadaveric controls without a previous history of urologic dysfunction had spontaneous contractile activity. This variability in the results could be explained by several factors such as the median age of the patients in our study, which was greater than that in the study by Mills et al.6 (63.3 versus 50.5 years, respectively) or the proportion of female versus male patients (5 women and 17 men in our study versus 8 women and 6 men in their study). In contrast, the mean resting tone of the spontaneous contractions and the occurrence of fused tetanic contractions were similar between the two studies. Although the myogenic nature of bladder phasic contractile activity has been clearly established in guinea pig13,14 and pig15 isolated bladder preparaUROLOGY 68 (2), 2006
FIGURE 2. Effect of glibenclamide on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip exposed to glibenclamide (1 and 10 M). (B) Effects of glibenclamide or vehicle on AUC of time-force curve, amplitude, and frequency of phasic contractions. Data are mean ⫾ SEM of three and five bladder samples for glibenclamide-treated and vehicle-treated conditions, respectively. Mean responses expressed as percentage of initial values measured before exposure to drug. P ⫽ NS, glibenclamide versus vehicle.
tions and shown in human detrusor in the present study, the mechanisms responsible for the occurrence of phasic mechanical activity have not yet been established. These phasic spontaneous events seem to be closely associated with spontaneous action potential. Spontaneous action potentials and associated calcium waves occur almost simultaneously along the boundary of bladder smooth muscle bundles and then propagate to the other boundary, probably through gap junctions.17 The investigation of electrical properties of human detrusor has revealed that calcium and potassium channels play an important role in the regulation of electrical activity of detrusor smooth muscle fibers.17 Thus, one may speculate that the participation of these channels in the regulation of human phasic contractile activity has been strongly suggested but never been clearly demonstrated. In human detrusor, L-type Ca2⫹ channels have been identified.18 Moreover, in guinea pig and pig isolated bladder strips, spontaneous phasic contractions are sensitive to the L-type Ca2⫹ channel inhibitors nisoldipine13,14 and nifedipine,15 respectively. Consistent with these data, we found that UROLOGY 68 (2), 2006
nifedipine suppressed human detrusor phasic contractions, suggesting that human detrusor phasic contractions also require extracellular Ca2⫹ entry through L-type Ca2⫹ channels. First, we investigated the participation of KATP channels in the regulation of human detrusor phasic contractions, because their participation has already been established in other animal species.15,19 We found no effect of the KATP channel inhibitor, glibenclamide, on phasic contractile activity, in accordance with data obtained from guinea pig19 and pig15 studies. This could suggest that KATP channels are closed and thus do not participate in the regulation of phasic contractions under basal conditions. KATP channels may rather be involved in the regulation of detrusor agonist-induced relaxation.20,21 In contrast, the KATP channel opener pinacidil dramatically reduced phasic contractile activity, and this effect was reversed by glibenclamide. Thus, one may speculate that the activation of KATP channels by KATP channel openers such as pinacidil22 or cromakalim15,23–25 may be sufficient to provoke membrane hyperpolarization, thereby altering smooth muscle cell contrac445
FIGURE 3. Effect of pinacidil and glibenclamide on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip displaying effect of pinacidil (10 M) on phasic contractions and restoring effect of glibenclamide (10 M). (B) Successive effects of pinacidil, glibenclamide, or their respective vehicles on AUC of time-force curve, amplitude, and frequency of phasic contractions. Data are mean ⫾ SEM of six and five bladder samples for drug-treated and vehicle conditions, respectively. Mean responses expressed as percentages of initial values measured before exposure to drugs. *P ⬍0.05, **P ⬍0.01, pinacidil versus vehicle; # P ⬍0.05, pinacidil versus glibenclamide; §P ⬍0.05, glibenclamide versus vehicle.
tility and disturbing the initiation of phasic contractions.15,26 Second, we also explored the participation of KCa (BK and SK) channels. The potent and selective blockers of the BKCa channel iberiotoxin and of SK2 and SK3 subtypes of the SKCa channel apamin markedly enhanced in vitro human bladder phasic contractions. Similar observations have been previously reported in isolated pig bladder.15 In addition, iberiotoxin has been reported to induce membrane depolarization and increase the frequency, amplitude, and duration of action potentials in guinea pig detrusor strips.27 Thus, the increase in phasic contractions induced by iberiotoxin or apamin we observed may have resulted from an increase of action potential amplitudes and/or frequencies and an enhancement of entry of Ca2⫹ through L-type Ca2⫹ channels. Also, studies with transgenic mice demonstrated a central role of BKCa and SKCa channels in urinary bladder function, particularly in spontaneous contractile activity.28,29 Thus, our results demonstrate the physiologic involvement of BKCa, SK2, and/or SK3 channels in the regulation of phasic contrac446
tile activity of the human detrusor. They could play prominent roles as negative-feedback elements to limit extracellular Ca2⫹ influx-mediated human detrusor phasic contractility. Additional studies investigating the effects of selective BKCa or SKCa channel openers are thus needed to better understand these mechanisms. Such selective openers might be expected to decrease human detrusor activity. Commercially available tools lack the selectivity required to test this hypothesis. CONCLUSIONS Pilot clinical studies performed in patients with OAB that tested first-generation K⫹ channel openers have revealed that these compounds may improve symptoms of urinary frequency and increase the mean voided volume; however, the benefit was limited by cardiovascular side effects.30,31 In the present study, we demonstrated that KATP and KCa channels participate in the regulation of human bladder myogenic phasic contractility. If myogenic spontaneous bladder contractions are involved in the origin of OAB, as previously suggested,2,8 uroUROLOGY 68 (2), 2006
FIGURE 4. Effect of iberiotoxin and apamin on human bladder strip phasic contractile activity. (A) Original tension recording developed by one detrusor strip exposed to iberiotoxin (100 nM). (B) Original tension recording developed by one detrusor strip exposed to apamin (100 nM). (C) Effects of iberiotoxin or apamin or vehicle on AUC of time-force curve, amplitude, and frequency of phasic contractions. Data are mean ⫾ SEM of five, six, and five bladder samples for iberiotoxin-treated, apamin-treated, and vehicle-treated conditions, respectively. Mean responses expressed as percentages of initial values measured before exposure to iberiotoxin, apamin, or vehicle. *P ⬍0.05, iberiotoxin versus vehicle and apamin versus vehicle.
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